Archive for March, 2009

You remember we had a competition, end of last year? Well, the runner-up of same – Dorian McIlroy – has claimed his prize by writing a guest blog. Clayton, that means you have to do two…?!

And Dorian writes on a subject close to our collective hearts, here in the Subunit Vaccine Group at Univ of CT: HIV vaccines, and how T-cell vaccines in particular may not be down and out after all. I note that he refers to two published papers that were topics of talks at the recent AIDS Vaccine Conference here at UCT recently, which was covered here in ViroBlogy: always useful to have your material on the verge of being published when you talk…!

HIV vaccine candidates – The return of the recombinants…

Towards the end of 2007 Merck interrupted a large scale clinical trial of a candidate HIV vaccine based on recombinant adenovirus. As well as showing no protective effect, there was a worrying tendency for vaccinees with serum antibodies to the vector – serotype 5 adenovirus (Ad5) – to show greater susceptibility to HIV infection than volunteers receiving placebo injection. This looked like the end of the road for adenovirus-based vaccines, and maybe even for any strategy based on recombinant viruses. However, two recent papers indicate that there may yet be considerable mileage in this approach.

The first, from Dan Barouch’s team in Boston, shows how recombinant adenoviruses could be made considerably more effective. One of the big drawbacks with these recombinant viruses is that repeated injections do not give an immunological booster effect. That is, even though the immune response to a single injection can be strong, there is not much increase after a second, or a third injection of the same recombinant virus. This happens because as well as inducing an immune response to the target antigen (SIV gag, for example), the recombinant virus vector induces an immune response against itself. So when the recombinant virus is injected a second time, it is neutralized by the antibodies induced by the first injection.

The Barouch paper shows that it is possible to get round this problem by using two recombinant adenoviruses with different serotypes, so that the second injection can provide an effective boost. Giving macaques two injections of SIV gag-recombinant Ad5 gave a good response to the first injection, but no boost. Using an SIV-gag recombinant Ad26 for the first injection, then using the Ad5 recombinant for the second, gave a 9-fold higher T-cell response measured by IFNgELISPOT than two doses of Ad5 recombinant.

So far, so good – but does this immune response translate to protection from infection?

Well, yes and no. After intravenous challenge with highly pathogenic SIVmac251, all animals in all groups were infected, so none of the animals were completely resistant to SIV. Peak viral load in the group vaccinated with Ad26/Ad5 was 1.4 log lower than in the control group, and about a log lower in the group vaccinated with Ad5/Ad5. Set-point viral load was also much lower in the Ad26/Ad5 group compared to the control group, so replication of the challenge virus was controlled to some extent in the animals that received the most effective vaccination protocol.

Overall there are two messages here. The first is that it is possible to do much better than the strategy that failed in the Merck trial, that employed three injections of Ad5 recombinant viruses. That’s the good news. The bad news is that even with a much more effective vaccination, in terms of the T-cell response, the protection against infection, although significant, was relatively modest. This means that vaccine candidates based on T-cell immunity alone are never going to work……. or does it?

Which brings me to the second paper, from Louis Picker’s group in Oregon. The big novelty here is the generation of recombinant rhesus macaque cytomegalovirus (CMV) expressing SIV proteins, that are tested as vaccine candidates in the SIV Mac model.

Why on earth would anyone want to try out CMV, when other recombinant (adeno- and pox-) viruses have been so disappointing? The difference is in the lifestyles of the viruses involved. Both adeno and poxviruses provoke acute infections, whereas CMV both in humans and apparently, in macaques, causes a lifelong infection, in which active viral replication is held in check largely by a robust T-cell response. When the immune response is compromised, as in AIDS patients, or transplant recipients receiving immunosuppressive drugs, CMV infection often reactivates, which can cause serious illness, and even be life-threatening. Because of the persistent nature of both the virus and the cellular immune response, a high-level of effector-memory T-cells (TEM, not to be confused with Transmission Electron Microscopy) specific for CMV are maintained in CMV+ individuals (who probably make up about half of the world population, in case you were wondering).

TEM have two interesting characteristics from a HIV vaccine point of view. Firstly, they are armed and dangerous. If they see their cognate viral antigen, they can either kill the infected cell, or secrete cytokines. Secondly, they migrate to mucosal sites, rather than lymph nodes, so they are in the right place to stop HIV infection after sexual transmission. More sedate central-memory T-cells (TCM), on the other hand, hang around the blood and lymph nodes, and do not immediately have anti-viral effector functions. Their response to HIV infection might be too late to be any use, as the first rounds of productive viral infection occur in the mucosa, not the draining lymph nodes.

Picker’s group points out that most memory T-cells that remain months after a recombinant adenovirus vaccination are TCM, not TEM, and set out to test the hypothesis that using a recombinant virus (CMV) that naturally gives a strong TEM response might be more effective in protecting against SIV infection at a mucosal site. So they generated recombinant, replication competent macaque CMV carrying genes for SIV gag, a Rev-Tat-Nef fusion protein, and an intracellular form of Env. Cells infected with these viruses expressed high levels of the recombinant proteins, and animals inoculated subcutaneously with them became persistently infected – just like wild-type CMV infection. As predicted, high levels of SIV-specific TEM were found in the blood, and in broncho-alveolar lavage (which is a relatively convenient way to obtain mucosal T-cells). More than one year after vaccination, animals were submitted to a mucosal challenge (intrarectal, if you really want to know) with SIVmac239, which is the same strain as that used to produce the recombinant CMV.

The details of the viral challenge are interesting. In order to simulate a real infection, a low viral dose was used, and this means that not all control animals get infected. So the challenge was repeated weekly until infection (detected by plasma viral load) was observed. I have turned the data from the paper into Kaplan-Meier curves, and used the log-rank test to compare the two groups (BTW: if Louis Picker is reading this, that’s the test you should have used). With p=0.03, survival without infection was significantly prolonged by vaccination, and 4 out of 12 vaccinated macaques were resistant to mucosal challenge. These four animals did not have a latent or cryptic infection, as no SIV DNA or RNA was detectable in CD4 T-cells in blood or lymph nodes, and CD8+ T-cell depletion did not result in viral rebound.

This was without any neutralizing antibodies, so for the first time a T-cell vaccination strategy has been shown to confer protection against infection (not just better control of viral load after infection, as with recombinant adenoviruses) in at least some vaccinated animals. Although 33% protection is not enough, at least things are going in the right direction.

So what’s the catch? Well, there are two really. Firstly the SIV sequences in the vaccine and challenge virus were identical. The vaccination strategy may not be so effective against a heterologous challenge, that would be more representative of the real-world. Secondly, the vaccination provokes a persistent infection with a genetically-modified virus, that (unlike other recombinant viruses and gene therapy vectors) remains infectious, so it’s hard to see how this kind of vaccine could be licensed for clinical trials.

Nevertheless, I think this paper is telling us – at last – what kind of T-cell response vaccines should be aiming to induce. Now all we need to do is solve the neutralizing antibody problem, and we’ll really be cooking with charcoal….

A fascinating new post from Science Daily – for which, thanks Vaibhav Bhardwaj – raises again the problem of just exactly what is (and what is not) a virus?

The article in question describes a fascinating three-way interaction between a virus, a parasitic wasp, and a caterpillar – with a virus which happens to be part of the germline of the wasp, gets injected into the caterpillar as a free genome, and modifies its immune system so as to tolerate the wasp’s eggs.

From the article:

“Researchers have known for about 40 years that some species of parasitoid wasps inject these viruses, known as polydnaviruses, into the body cavities of caterpillars at the same time that they lay their eggs in the caterpillars. Because these “virus-like particles” have become an integral part of the wasp genome, some researchers have suggested they should no longer be considered viruses.”

So there is ample precedent for things that integrate into host genomes, that are also viruses – including, among vertebrates, where activatable endogenous retroviruses are the subject of much study – including a finding that prions may activate endogenous retroviruses in the human brain.

And what of polydnaviruses (family Polydnaviridae)? Well, fascinating and unique beasts, these: the two genera so far described – Bracovirus and Ichnovirus – contain viruses which have a variable number of circular double-stranded DNA components, with components ranging in size from 2 to >31 kbp, for a total genome size between 150 – 250 kbp. Both sets of viruses occur as integrated proviruses in the genomes of endoparasitic hymenopteran wasps, replicate by amplification of the host DNA, followed by excision of episomal genomes by site-specific recombination, and only produce particles by budding from (ichnoviruses) or lysis of (bracoviruses) calyx cells in the oviducts of female wasps during pupal-adult transition. Moreover, the viruses in the two groups may well not be evolutionarily linked to one another, given that there is no antigenic or genome similarity, and the particles formed by the two groups are very different: ichnoviruses make ellipsoidal particles with double membranes containing one nucleocapsid; bracoviruses make single-enveloped particles containing one or more cylindrical nucleocapsids. The latter may derive from nudiviruses, which appear to have contributed very substantially to wasp survival.

“What this means… is that nudiviruses infected wasps a few million years ago and that, over time, the viral DNA fully integrated into the wasp genome. As it currently stands, the wasps need the virus to survive, because the virus helps the insects lay eggs in caterpillars. The virus also needs the wasp to survive, because the virus can only replicate in the wasp’s ovaries. The virus cannot replicate inside the caterpillar, because all of its replication machinery is inside the wasp.”

Particles are injected along with eggs into larvae of lepidopteran hosts; the DNA gets into secondary host cells and is expressed, but does not replicate -and this expression leads to some quite profound phsiological changes, many of which are responsible for successful parasitism. The association between wasp and virus has been termed an “obligate mutualistic symbiosis”, and appears to have evolved over more than 70 million years. It does nothing for the lepidopterans, however….

But nothing in this association would lead me to doubt their nature as viruses. Viruses now dependent on a particular host, maybe, but there now appears to be a continuum in the virus life experience, from massive bigger-than-cell-genome mimiviruses, through parasitic viruses to tiny circoviruses – and including agents that have become irretrievably integrated into the genomes of other hosts, yet still have a partially independent existence, like pseudoviruses – and polydnaviruses.